The major objective of the proposed project is to develop methodology that will allow fast and efficient detection of infectious prion proteins prior to the onset of clinical symptoms. This includes the detection of infectious prions in mortal products, early ante-mortem diagnostic of the prion diseases, and developing experimental approaches allowing delineation of the molecular mechanism of prion and prion-related diseases (folding disorders) in thermodynamic and kinetic terms. Preliminary studies successfully demonstrated that the specific detection of the pathologic prion protein could be approached by simple experimental means. The prion disease process involves a conformational change in the prion protein that in turn can serve as the basis for an early detection, diagnostic, prognostic and screening assay. The assay detects infectivity in a crude sample by identifying existing sub-picomolar levels of infectious prion protein through the use of specific, fluorescent target peptides. The labeled peptides undergo conformational change resulting in an amplified signal in a single step without protease pre-treatment, denaturants or washing. This methodology has three applications: first as a detection assay for infectious prion protein, second as a possible tool for similar diagnostics in other amyloid and/or mis-folded protein diseases; and, lastly as a tool for gaining a better understanding of the molecular mechanism of this group of diseases which can be used to optimize diagnostics and future therapeutics. The structural complexity of the target peptide sequence will be optimized and the thermodynamic and kinetic facets of peptide interaction with the infectious prion protein will be studied thoroughly at the quantitative level to highlight the prominent features that determine species specificity and to generate calibration curves with binding constants for diagnostic development. To further assay improvement, a predictive model will be developed for the conformational diversity of the target peptides used as indicators and surrogates of conformational change upon interaction with the infectious prion protein. The universality of the target peptide concept as a detector for other amyloid diseases will also be evaluated.